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V o l . 4 0 BOVINE CERVICAL MUCINS 3 3 7REFERENCES

E l s o n , L . A . & M o r g a n , W . T . J . ( 1 9 3 3 ) . B i o c h e m . J . 2 7 , 1 8 2 4 .G o l d s w o r t h y , N . E . & F l o r e y , H . ( 1 9 3 0 ) . B r i t . J . e x p . P a t h .1 1 , 1 9 2 .H a g e d o r n , H . C . & J e n s e n , B . N . ( 1 9 2 3 ) . B i o c h e m . Z . 1 3 5 , .4 6 .H a l e , C . W. ( 1 9 4 4 ) . B i o c h e m . J . 3 8 , 3 6 2 .K i n g , E . J . ( 1 9 3 2 ) . B i o c h e m . J . 2 6 , 2 9 2 .M e h l , J . W. ( 1 9 4 5 ) . J . b i o l . C h e m . 1 5 7 , 1 7 3 .M o r g a n , W. T . J . & E l s o n , L . A . ( 1 9 3 4 ) . B i o c h e m . J . 2 8 , 9 8 8 .

R i m i n g t o n , C . ( 1 9 4 0 ) . B i o c h e m . J . 3 4 , 9 3 1 .S a c k e t t , G . E . ( 1 9 2 5 ) . J . b i o l . C h e m . 6 4 , 2 0 3 .S c o t t B l a i r , G . W . , C o w i e . A . T . & F o l l e y , S . J . ( 1 9 4 2 ) .J . P h y s i o l . 1 0 1 , 1 1 P .S c o t t B l a i r , G . W . , F o l l e y , S . J . , M a l p r e s s , F . H . & C o p p e n ,F . M . V . ( 1 9 4 1 ) . B i o c h e m . J . 8 5 , 1 0 3 9 .S t a c e y , M . ( 1 9 4 3 ) . C h e m . & I n d . 6 2 , 1 1 0 .W o o d m a n , H . E . & Hammond, J . ( 1 9 2 5 ) . J . a g r i c . S c i . 1 5 ,1 0 7 .

S t u d i e s i n P e n i c i l l i n P r o d u c t i o n b y P e n i c i l l i u m n o t a t u m ;i n S u r f a c e C u l t u r e1 . A PRELIMINARY STUDY OF THE METABOLISM OF CARBON,

NITROGEN, SULPHUR AND PHOSPHORUS- B y D . J . D . HOCKENHULL, I m p e r i a l C h e m i c a l I n i u s t r i e s L t d . , R e 8 e a r c h L a b o r a t o r i e 8 ,

B l a c k l e y , M a n c h e 8 t e r -( R e c e i v e d 1 6 J a n u a r y 1 9 4 6 )

The f o l l o w i n g s t u d i e s o n P e n i c i l l i u m n o t a t u m w e r ei n t e n d e d t o p r e p a r e t h e wa y f o r a more d e t a i l e di n v e s t i g a t i o n o f t h e method by w h i c h t h e mouldp r o d u c e s p e n i c i l l i n , and f u r t h e r m o r e t o a s c e rt ai n t owhat e x t e n t t h e p r o d u c t i o n o f p e n i c i l l i n c o u l d b ei n c r e a s e d by c o n t r o l l i n g t h e c u l t u r a l e n v i r o n m e n t .

I t i s i m p o r t a n t t o n o t e t h a t t h e p r o d u c t i o n o fp e n i c i l l i n o n l y r e p r e s e n t s a m i n u t e f r a c t i o n o f t h et o t a l m e t a b o l i c a c t i v i t y o f t h e m o u l d , i n c o n t r a s tt o t h e p r o d u c t i o n o f c i t r i c a c i d by A 8 p e r g i l l u 8 n i g e r .M o r e o v e r , n o n e o f t h e common i n t e r m e d i a t e p r o -d u c t s o f m e t a b o l i s m s u c h a s o x a l i c , f u m a r i c a n dg l u c o n i c a c i d s o r g l y c e r o l , e t c . , c o u l d b e d e t e c t e di n o t h e r t h a n n e g l i g i b l e q u a n t i t i e s d u r i n g t h e g r o w t ho f P e n i c i l l i u m n o t a t u m u n d e r c o n d i t i o n s f a v o u r i n gp e n i c i l l i n p r o d u c t i o n . I t t h u s a p p e a r e d t h a t a ne s s e n t i a l p r e l i m i n a r y t o i n v e s t i g a t i o n s o f t h e m e c h a -n i s m o f p e n i c i l l i n p r o d u c t i o n wa s t o a s c e r t a i n t h ef a t e o f i n d i v i d u a l c o n s t i t u e n t s o f t h e c u l t u r em e d i u m . I n t h e p r e s e n t i n v e s t i g a t i o n a n a t t e m p th a s b e e n m a d e t o f o l l o w t h e c h a n g e s u n d e r g o n e bysome o f t h e p r i n c i p a l n u t r i e n t s u b s t a n c e s d u r i n gt h e g r o w t h o f t h e m o u l d .E x i s t i n g m e t h o d s o f a n a l y s i s h a v e b e e n m o d i f i e dt o d e a l w i t h t h e s u b s t a n c e s i n v e s t i g a t e d .

EXPERIMENTALC u l t u r a l m e t h o d s a n d m a t e r i a l s

C o M p o s i t i o n o f m e d i u m . The e a r l i e r p a r t o f t h ework wa s c a r r i e d o u t u s i n g a m o d i f i e d C z a p e k -

T u r l e y medium s u b s e q u e n t l y r e f e r r e d t o a s c a e i r nmedium o f w h i c h t h e c o m p o s i t i o n w a s :G l u c o s e ( f i r s t g r a d e c o m m e r c i a l q u a l i t y ) 3NaNO3 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 3K H 2 P O 4 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 . 1KC1 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 0 5M g S O 4 . 7 H M 2 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 - 0 2 5F e S O 4 . 7H20 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 0 0 1C a s e i n , a s 2 0 % ( v / v ) H O E d i g e s t 0 * 5

T h i s medium was m a d e up i n t a p w a t e r and a d -j u s t e d t o pH 4 - 5 w i t h 4 0 % NaOH b e f o r e s t e r i l i z a -t i o n .

I t was communicated t o u s by Dr C o g h i l l o fP e o r i a , U . S . A . , t h a t p e n i c i l l i n p r o d u c t i o n wasg r e a t l y i n c r e a s e d by t h e u s e o f c o rn - s t e e p l i q u o r( 3 0 % t o t a l s o l i d s ) i n t h e medium. A c c o r d i n g l y amedium, known a s CSL medium was a d o p t e d f o rc o m m e r c i a l p r o d u c t i o n o f p e n i c i l l i n . T h i s me diumwas c o n s e q u e n t l y u s e d i n t h e i n v e s t i g a t i o n . I t sc o m p o s i t i o n w a s :L a c t o s e B . P . 3G l u c o s e , c o m m e r c i a l ( a s a b o v e ) 1F i l t e r e d c o r n - s t e e p l i q u o r 8N a N O 3 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 3K H , P 0 4 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 01M g S 0 4 . 7 H 2 0 ( a n a l y t i c a l r e a g e n t q u a l i t y ) . 0 - 0 2 5Z n S O 4 . 7 H 2 0 ( a n a l y t i c a l r e a g e n t q u a l i t y ) ( - 0 0 1M n S O 4 . 4 H 2 0 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 - 0 0 1C u S O % . 5 H 2 0 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 0 0 1F e S O 4 . 7 H 2 0 ( a n a l y t i c a l r e a g e n t q u a l i t y ) 0 0 0 1T h i s medium was made up i n t a p w a t e r and a d -j u s t e d t o p H 4 - 5 w i t h 4 0 % ( w / v ) NaOH b e f o r es t e r i l i z a t i o n .

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D . J . D . HOCKENHULLS t r a i n o f o r g a n i . m . At t h e same t i m e a s t h ec h a n g e t o CSL m e d i u m , t h e o r i g i n a l s t r a i n used ont h e c a s e i n m e d i u m and o b t a i n e d from S i r Howar dF l o r e y and d e s c r i b e d h e r e a s ' s t r a i n F 4 ' , was sup-p l a n t e d by t h e more e f f i c i e n t - p e n i c i l l i n - p r o d u c i n gs t r a i n o b t a i n e d f r o m t h e N o r t h e r n R e g i o n a l R e -s e a r c h L a b o r a t o r i e s , P e o r i a , I l l i n o i s , and k n ow n a s

s t r a i n 1 2 4 9 B 2 1 .G r o w t h c o n d i t i o n 8 . E x p . 1 was c a r r i e d o u t i ne a r t h e n w a r e c u l t u r e v e s s e l s a s d e s c r i b e d by C h a i n ,F l o r e y , G a r d n e r , H e a t l e y , J e n n i n g s , O r r - E w i n g &S a n d e r s ( 1 9 4 1 ) . One l i t r e o f m e d i u m g i v i n g a d e p t ho f I i n . was u s e d i n e a c h v e s s e l .

were i n c u b a t e d a t 2 3 - 5 + 0 . 5 0 t h r o u g h o u t . U n l e s so t h e r w i s e s t a t e d a l l e x p e r i m e n t a l b o t t l e s were s e tup i n d u p l i c a t e .

M e t h o d 8 o f a n a l y 8 i 8T o t a l c a r b o n b y w e t c o m b u t i o n . T h e s a m p l e , c o n t a i n i n g5 - 8 mg. c a r b o n , was d r i e d down a t 1 1 0 0 a n d o x i d i z e d b y

a c h r o m i c - s u l p h u r i c a c i d m i x t u r e a n d t h e C 0 0 e v o l v e dc o l l e c t e d i n 0 - 0 5 M - b a r y t a i n a s p e c i a l s p i r a l b u b b l e r a f t e rP e a r c e &R o b e r t s o n ( 1 9 3 6 ) . T h e m e t h o d i s a m o d i f i c a t i o no f t h a t o f M c C r e a d y &H a s s i d - ( 1 9 4 2 ) .R e d u c i n g s u g a r a 8 g l u c o 8 e . I t was n o t f o u n d n ec e s s a r yt o c l e a r t h e f i l t e r e d medium b e f o r e a n a l y s i s . Medium wash y d r o l y z e d f o r 1 h r . - w i t h an e q u a l v o l u m e o f 2 N - H C 1 ,0E0I~w. -

A . * ..-..,-_... '' ? '

C H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

Fig. 1. Aerated-culture apparatus with gas-sampling device. A, soda-lime tube; B, air humidifier; C, pressureregulator;D, release va lv e; E , B air s t e r i l i z e r (co t t o n w o o l ) Fculture f l a s k ; manometric f l o w m e t e r ; Hgassamplngv e s s e l ; T^,air iulet tube; T.,air exit tube; T3 sampling tube K, 25ml fla s k, hermetic seal for T., P , cottonwool p l u g in T, .

A l l s u b s e q u e n t e x p e r i m e n t s were c a r r i e d o u t i ng l a s s c u l t u r e v e s s e l s a s d e s c r i b e d by C l a y t o n , H a n e s ,R o b i n s o n , Andrews &Hunwicke ( 1 9 4 4 ) ; 5 0 0 m l . o fm e d i u m / v e s s e l gave c . I i n . l a y e r .Steam s t e r i l i z a t i o n was c a r r i e d on f o r 2 0 m i n . a t1 5 l b . / s q . i n . i n a l l e x p e r i m e n t s .Th e i n o c u l u m was p r e p a r e d a s f o l l o w s : A c u l t u r eo f t h e mould on m i n c e d p o t a t o ( t w i c e s t e r i l i z e d a t1 2 0 l h . / s q . i n . f o r 2 0 m i n . ) grown f o r 5 d a y s and t h e ns h a k e n w i t h a 0 1%o l u t i o n o f a w e t t i n g a g e n t' C a l s o l e n e o i l HS' ( I . C . I . , L t d . ) . Th e r e s u l t a n t s p o r es u s p e n s i o n was s p r a y e d i n t o t h e . c u l t u r e v e s s e l s bymeans o f a s t e r i l e ' A e r o g r a p h ' s p r a y gu n . T h i s p r o -c e d u r e gave r a p i d and even c o v e r a g e . A l l c u l t u r e s

n e u t r a l i z e d a n d d i l u t e d so a s t o c o n t a i n t h e e q u i v a l e n t o f1 - 1 - 5 mg. g l u c o s e i n a s a m p l e o f 5 m l . The g l u c o s e e q u i v a l e n to f t h e s o l u t i o n was d e t e r m i n e d b y a m o d i f i c a t i o n o f t h em e t h o d o f S o m o g y i ( 1 9 3 7 ) d u e t o H a n e s ( H o c k e n h u l l &H e r b e r t , 1 9 4 5 ) .

C a r b o n d i o x i d e . S a m p l e s o f ga s f r o m c o n t i n u o u s l y a e r a t e db o t t l e s f i t t e d w i t h f l o w m e t e r ( F i g . 1 ) were a n a l y z e d b ya b s o r b i n g t h e C 0 2 i n 0 - 0 5 N - b a r y t a . A s p i r a l b u b b l e r s i m i l a rt o t h a t u s e d i n t h e method f o r t o t a l c a r b o n was u s e d .

T o t a l n i t r o g e n . S a m p l e s o f medium o r m y c e l i u m con-t a i n i n g a b o u t 2 mg. N , were d r i e d a t 1 1 0 0 a n d a n a l y z e db y t h e m o d i f i e d K j e l d a h l p r o c e d u r e o f P e p k o w i t z &S h i v e( 1 9 4 2 ) .N i t r a t e . . A f i l t e r e d s o l u t i o n o f t h e medium c o n t a i n i n g3 - 5 m g. o f n i t r o g e n a s n i t r a t e was d i l u t e d t o 2 0 m l . , a c i d i f i e d

3 3 8 I 9 4 6

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D a y sF i g . 3 .F i g . 2 . C a r b o n m e t a b o l i s m o f P . n o t a t u m ; e a r t h e n w a r e c u l t u r e v e s s e l s . E x p . 1 , s t r a i n F 4 , c a s e i n m e d i u m , 1 1 . / v e s s e l .

o =t o t a l c a r b o n i n m e d i u m ; * = c a r b o n a s s u g a r ( g l u c o s e e q u i v a l e n t s ) ; o = c a r b o n i n m y c e l i u m ; a = p e n i c i l l i n t i t r e .F i g . 3 . C a r b o n m e t a b o l i s m o f P . n o t a t u m ; i n g l a s s c u l t u r e - f l a s k s . E x p . 2 , s t r a i n F 4 , c a e e i n m e d i u m , 5 0 0 m l . / v e s s e l .o = t o t a l c a r b o n i n m e d i u m ; * = c a r b o n a s s u g a r ( g l u c o s e e q u i v a l e n t s ) ; o = c a r b o n l o s t a s C0; a = p e n i c i l l i n t i t r e . .

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D a y s D a y sF i g . 4 . F i g . 5 .F i g . 4 . C h a n g e s i n p H v a l u e s b y t w o s t r a i n s o f P . n o t a t u m . E x p . 1 , s t r a i n F 2 , c a 8 e i n m e d i u m ; E x p . 3 , s t r a i n 1 2 4 9 ,CSL m e d i u m . o= p H v a l u e s , s t r a i n F 4 ; * = p e n i c i l l i n t i t r e s , s t r a i n F 4 ; o =p H v a l u e s , s t r a i n 1 2 4 9 ; * = p e n i c i l l i nt i t r e s , s t r a i n 1 2 4 9 .F i g . 5 . C a r b o n m e t a b o l i s m o f P . n o t a t u m . E x p . 3 , s t r a i n 1 2 4 9 , CSL m e d i u m . o = t o t a l c a r b o n i n m e d i u m ; * = c a r b o n a ss u g a r g l u c o s e e q u i v a l e n t s ; * = p e n i c i l l i n t i t r e .

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D . J . D . HOCKENHULLw i t h 0 -5 m l . conc. H 2 S 0 4 a n d t h e n i t r a t e p r e c i p i t a t e d w i t hexce s s o f a 1 0 % s o l u t i o n o f ' n i t r o n r e a g e n t ' , a n d d e t e r -m i n e d g r a v i m e t r i c a l l y a f t e r w a s h i n g w i t h i c e - c o l d w a t e ra n d d r y i n g a t 1 1 0 0 .A m i n o - n i t r o g e n . T h e medium was a n a l y z e d f o r a m i n o -n i t r o g e n b y t h e m e t h o d o f P o p e & S t e v e n s ( 1 9 3 9 ) . N e i t h e ro f t h e m e d i a u s e d c o n t a i n e d i n t e r f e r i n g s u b s t a n c e s .V o l a t i l e - b a s e n i t r o g e n . A s a m p l e o f medium c o n t a i n i n g0 - 4 m g. o f n i t r o g e n a s v o l a t i l e b a s e was steam d i s t i l l e df r o m 1 0 m l . s a t . s o l u t i o n o f N a 2 C O , a n d t h e d i s t i l l a t e c o l -l e c t e d i n 0 - 1 N - H C I , w h i c h was b a c k - t i t r a t e d a s i n t h em e t h o d a b o v e f o r K j e l d a h l n i t r o g e n .P h o s p h a t e . F r e e o r t h o p h o s p h at e was e s t i m a t e d b y t h em e t h o d o f Y o u n g b e r g &Y o u n g b e r g ( 1 9 3 0 ) . C o l o u r com-p a r i s o n was made i n a H i l g e r ' S p e k k e r ' a b s o r p t i o m e t e ru s i n g an o r a ng e f i l t e r .T o t a l s u l p h u r . T h e s a m p l e , c o n t a i n i n g c . 0 - 5 . m g . o fs u l p h u r , was d r i e d down a t 1 1 0 0 a n d o x i d i z e d w i t h a n i t r i ca c i d - b r o m i n e m i x t u r e i n w h i c h h a d b e e n d i s s o l v e d z i n co x i d e . T h e c o m p l e t e l y o x i d i z e d s u l p h u r , a s z i n c s u l p h a t e ,was r e d u c e d w i t h a h y d r i o d i c - h y p o p h o s p h o r o u s a c i d m i x -t u r e t o H 2 S , w h i c h was d i s t i l l e d o f f a n d c o l l e c t e d i n NaOH.S t a n d a r d i o d i n e was a d d e d a n d t h e s o l u t i o n a c i d i f i e d , t h eu n u s e d i o d i n e b e i n g t i t r a t e d w i t h t h i o s u l p h a t e . T h e m e t h o da n d a p p a r a t u s a re m o d i f i e d f r o m t h o s e u s e d b y ' L u k e ( 1 9 4 3 ) .P e n i c i l l i n . The p e n i c i l l i n t i t r e s o f t h e m e d i a were d e t e r -m i n e d b y my colleague, M r s I . F . H o g g a r t h , b y a m e t h o ds i m i l a r t o t h a t o f F o s t e r &W o o d r u f f ( 1 9 4 3 ) . F i g u r e s q u o t e da r e in O x f o r d u n i t s ( o . u . ; A b r a h a m , C h a i n , F l e t c h e r ,F l o r e y , G a r d n e r , H e a t l e y & J e n n i n g s ( 1 9 4 1 ) ) . A b e r r a n tp o i n t s o c c u r on c e r t a i n o f t h e f i g u r e s . T h e s e a r e d u e t oa b r e a k d o w n o f t h e a s s a y s y s t e m on t h e d a y s c o p c e r n e d .

C a r b o n m e t a b o l i s mU s i n g s t r a i n F4 . I n E x p . 1 , s i x t y r e p l i c a t ee a r t h e n w a r e b o t t l e s were u s e d a s d e s c r i b e d a b o v e .

B o t t l e s were removed a t 2 4 h r . i n t e r v a l s f o r d e t e r -m i n a t i o n o f t h e r e d u c i n g sugar, t o t a l c a r b o n andp e n i c i l l i n c o n c e n t r a t i o n s i n t h e m edi u m and t h et o t a l c a r b o n i n t h e m y c e l i u m . A f t e r an i n i t i a l l a gp h a s e b o t h t h e c a r b o n and t h e sugar f e l l s t e a d i l ya s shown i n F i g . 2 . The mycelium c a r b o n a f t e r ani n i t i a l l a g p h a s e r o s e t o a m a x i m u m on t h e 1 0 t h dayand then f e l l g r a d u a l l y ( F i g . 2 ) . T h e r e was a s t e a d yr i s e i n pH v a l u e t h r o u g h o u t ( F i g . 4 ) .E x p . 2 was c a r r i e d out i n g l a s s c u l t u r e v e s s e l sa s d e s c r i b e d a b o v e . Th e v e s s e l s were f i t t e d up sot h a t t h e r a t e o f a e r a t i o n and of CO2 p r o d u c t i o nc o u l d b e m e a s u r e d ( F i g . 1 ) . D e t e r m i n a t i o n o f r e -d u c i n g su gar , t o t a l c a r b o n and p e n i c i l l i n concentra-t i o n s i n t h e m ed iu m, a nd t h e r a t e o f e v o l u t i o n o fCO2 were m a d e d a i l y ( F i g . 3 ) . Th e m y c e l i a l c a r b o nwas d e t e r m i n e d a t t h e end o f t h e f e r m e n t a t i o n ;9 8 % f t h e c a r b o n was a c c o u n t e d f o r .U s i n g s t r a i n 1 2 4 9 . As m e n t i o n e d a b o v e , CSLmedium was u s e d t h r o u g h o u t w i t h s t r a i n 1 2 4 9 . Ont h i s medium, w h i c h c o n t a i n e d more c a r b o n andn i t r o g e n t h a n d i d t h e c a s e i n medium, p e n i c i l l i ny i e l d s were m uc h g r e a t e r t h a n t h o s e o b t a i n e d withs t r a i n F4 ( 8 0 - 1 2 0 o . u . / m l . as compared w i t h 20-2 5 o . u . / m l . w i t h t h e l a t t e r ) .

I n E x p . 3 , t o t a l c a r b o n and r e d u c i n g s uga r wered e t e r m i n e d ( F i g . . 5 ) and showed t h e same grosse f f e c t s a s w i t h s t r a i n F 4 ; t h e pH c h a n g e s were a l s overy s i m i l a r ( F i g . 4 ) .I n E x p . 3 , t h e CO2 o u t p u t was r e c o r d e d a s inExp. 2 . I t was shown t h a t t h e p e a k o f r e s p i r a t o r ya c t i v i t y p r e c e d e d t h e p e a k o f p e n i c i l l i n c o n c e n t r a -t i o n by a b o u t 2 d a y s ( F i g . 6 ) .

. 55.

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D a y sF i g . 6 . R e s p i r a t i o n o f P . n o t a t u m . E x p . 4 , s t r a i n 1 2 4 9 ,CSL m e d i u m . o = r a t e o f C O 2 e v o l u t i o n ; * = p e n i c i l l i nt i t r e .

N i t r o g e n m e t a b o l i s mU s i n g s t r a i n F4 . No e x p e r i m e n t s on amino-n i t r o g e n u t i l i z a t i o n o r v o l a t i l e b a s e p r o d u c t i o n were

c a r r i e d o u t w i t h t h i s s t r a i n . I n E x p . 1 , t o t a ln i t r o g e n and n i t r a t e n i t r o g e n i n t h e medium, t o t a lm y c e l i a l n i t r o g e n and p e n i c i l l i n were d e t e r m i n e d ,a t 2 4 h r . i n t e r v a l s ( F i g . 7 ) . A f t e r a v e r y s h o r t l a gp h a s e , b o t h t o t a l n i t r o g e n and n i t r a t e n i t r o g e n f e l lr a p i d l y . Th e t o t a l n i t r o g e n f e l l , by t h e 1 1 t h d a y ,t o a m i ni m u m v a l u e o f a b o u t 3 0 %o f i t s i n i t i a ll e v e l ; t h e n i t r a t e n i t r o g e n f e l l , a s y m p t o t i c a l l y , t ozero on t h e 1 4 t h d a y . P e n i c i l l i n p r o d u c t i o n l a g g e db e h i n d t h e s e c u r v e s by a b o u t 3 d a y s . A f t e r t h e1 1 t h d ay , t o t a l n i t r o g e n i n t h e m e d i u m i n c r e a s e dby t h e 1 4 t h day t o a b o u t 5 5 % f i t s i n i t i a l v a l u e .Th e u p t a k e o f n i t r o g e n from t h e m e d i u m was re -f l e c t e d by t h e i n c r e a s e i n m y c e l i a l n i t r o g e n whichr e a c h e d i t s m a x i m u m a t a b o u t t h e same t i m e a ndt h e n d e c r e a s e d , r e f l e c t i n g t h e r e a p p e a r a n c e o f n i t r o -gen i n t h e medium. Th e d i s c r e p a n c y between thetwo s e r i e s was due t o t h e m e t h o d o f r e p l i c a t esampling.U s i n g s t r a i n 1 2 4 9 . In E x p . 1 5 , s i x r e p l i c a t eb o t t l e s were e x a m i n e d . CSL medium was e m p l o y e d .T o t a l n i t r o g e n and a m i n o - n i t r o g e n i n t h e m e d i u mand p e n i c i l l i n were d e t e r m i n e d a t 2 4 h r . i n t e r v a l s

3 4 0 I 9 4 6

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V o l . 4 0 METABOLISM OF PEN( F i g . 8 ) . A f t e r i n i t i a l l a g s t h e a m i n o - n i t r o g e n c o n -c e n t r a t i o n f e l l s t e a d i l y t h r o u g h o u t t h e m e t a b o l i s mp e r i o d . The t o t a l n i t r o g e n c u r v e t e n d e d t o f l a t t e no u t by t h e 1 0 t h d a y ; i t d i d n o t , h o w e v e r , show amarked minimum l i k e t h a t o b s e r v e d i n Exp. 1( F i g . 7 ) , a l t h o u g h n i t r o g e n was u t i l i z e d t o a b o u tt h e same e x t e n t .

1 - 2 4 8. 4 61 - 1 4 44 21 - 0 4 03 80 - 9 - - 3 6- 3 46-s~~~~~~~~~~~~~-32

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F i g . 7 . N i t r o g e n m e t a b o l i s m o f P . n o t a t u m . E x p . 1 ,s t r a i n F 4 , c a s e i n m e d i u m , 1 L . / v e s s e l . o = t o t a l n i t r o g e ni n m e d i u m ; * = n i t r a t e n i t ro g e n i n m e d i u m ; o = n i t r o g e ni n m y c e l i u m ; = p e n i c i l l i n t i t r e .I n E x p . 6 , two s e t s , A and B , t e n r e p l i c a t e b o t t l e sw e r e s e t u p , sown a n d grown i n t h e n o r m a l wa yu n t i l t h e 8 t h d a y o f g r o w t h . At t h i n p o i n t , 4 - 7 g .( N H 4 ) 2 S 0 4 , A . R . d i s s o l v e d i n 2 0 m l . o f w a t e r a n ds t e r i l i z e d w e r e c a r e f u l l y added t o t h e m edi u m i ne a c h b o t t l e i n s e t B ; t h i n a d d i t i o n c o r r e s p o n d e d t o

2 m g . N / m l . a s a m m o n i u m s a l t . 2 0 m l . o f s t e r i l ew a t e r w a s a d d e d t o e a c h b o t t l e o f t h e c o n t r o l s e t A .

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s t r a i n 1 2 4 9 , CSL m e d i u m . o = t o t a l n i t r o g e n i n m e d i u m ;* =amino n i t r o g e n i n m e d i u m ; * = p e n i c i l l i n t i t r e .

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F i g . 9 . V o l a t i l e - b a s e p r o d u c t i o n a n d p e n i c i l l i n y i e l d o fP . n o t a t u m . E x p . 6 , s t r a i n 1 2 4 9 , CSL m e d i u m . o =v o l a t i l e - b a s e n i t r o g e n i n m e d i u m ; * = p e n i c i i l i n t i t r e .T a b l e 1 . T h e e f f e c t o f t h e a d d i t i o n o f a m m o ni u m i o n t oc u l t u r e s o f P e n i c i l l i u m notatum

P e n i c i l l i n t i t r e( m e a n o f 1 0 ) o . u : / m l .A 2 m g . N / m l .No a d d e d a d d e d a s[ N H 4 ] + [ N H 4 ] +

8 3 - 9 7 8 - 37 5 - 2 7 9 - 29 2 - 8 6 8 - 89 0 - 0 5 6 - 57 6 - 1 5 7 - 53 6 - 3 1 1 - 6

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D . J . D . HOCKENHULLI n t h e c o n t r o l s e t A , t h e v o l a t i l e b a s e n i t r o g e nr e m a i n e d l o w and s t e a d y a r o u n d 0 - 2 5 m g . N / m l .

u n t i l t h e 8 t h day when i t r o s e s t e ad i l y t o t h e l e v e lo f 1 - 0 5 m g . N / m l . o n t h e 1 6 t h d a y . T h i s wa s a c c o m -p a n i e d by a d e c l i n e i n p e n i c i l l i n t i t r e ( F i g . 9 ) . Thed e c l i n e i n p e n i c i l l i n t i t r e w a s , h o w e v e r , f a r g r e a t e ri n s e r i e s B w h i c h c o n t a i n e d a d d e d a m m o n i u m i o n( T a b l e 1 ) . S i n c e t h e pH v a l u e s r e c o r d e d f o r e a c hs e r i e s w e r e t h e s a m e , i t s e e m s l i k e l y t h a t t h e [ N H 4 ] +i o n c au s e s t h i s a c c e l e r a t i o n i n t h e d e s t r u c t i o n o fp e n i c i l l i n a t pH v a l u e s a b o v e 7 - 5 .

P h o s p h o r u s m e t a b o l i s mS t r a i n 1 2 4 9 and CSL medium w e r e u s e d f o r t h i ss e r i e s o f e x p e r i m e n t s .I n E x p . 7 , w h i c h w a s d o n e i n s i x r e p l i c a t e b o t t l e s ,t h e p h o s p h a t e and p e n i c i l l i n i n t h e medium w e r ed e t e r m i n e d a t 2 4 h r . i n t e r v a l s . The p h o s p h a t e l e v e l

( 9 0 - 1 0 0 o . u . / m l . ) . Th e p h o s p h a t e m e t a b o l i s m f o l -l o w e d a c o u r s e s i m i l a r t 6 t h a t o f E x p . 7 .I n E x p . 9 a medium c o n t a i n i n g o n l y 0 0 4 mg.P / m l . was u s e d . T h i s was p r e p a r e d by t h e methodu s e d i n E x p . 8 . W h e n t h e m y c e l i a l f e l t had b e e nf u l l y e s t a b l i s h e d ( 5 d a y s ) , s t e r i l e a d d i t i o n s o f s o d i u mi o d o a c e t a t e , s o d i u m f l u o r i d e o r s o d i u m a r s e n a t e a t0 1 m g . / m l . o f medium o r s o d i u m s e l e n i t e a t 0 . 1and 1 - 0 m g . / m l . o f m edi u m w e r e m a d e . None o ft h e s e i n h i b i t o r s a t 0 * 1 m g . / m l . had any e f f e c t onp e n i c i l l i n y i e l d . H o w e v e r , 1 0 m g . / m l . o f s o d i u ms e l e n i t e c u t down p e t r i c i l l i n y i e l d t o 5 0 % o f t h en o r m a l .

S u l p h u r m e t a b o l i s mS t r a i n 1 2 4 9 was u s e d i n E x p . 1 0 . Th e m edi u mwas a n o r m a l CSL medium e x c e p t t h a t t h e c o r n -

s t e e p l i q u o r had b e e n p r e t r e a t e d by e x t r a c t i o n w i t hh o t b u t a n o l c o n t i n u o u s l y f o r 1 2 h r . a t p H 3 7 - 3 9 ;

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D a y sF i g . 1 0 . P h o s p h a t e m e t a b o l i s m o f P . n o t a t u m . E x p . . 5 ,s t r a i n 1 2 4 9 , CSL m e d i u m . o = p h o s p h a t e p h o s p h o r u s i nm e d i u m ; * = p e n i e i l h i n t i t r e .s h o w e d c o n s i d e r a b l e c h a n g e s . I t f e l l t o a m i ni m u mon t h e 4 t h d a y , by w h i c h t i m e a t h i c k m y c e l i a l math a d f o r m e d . A f t e r t h i s i t r o s e a l m o s t t o i t s i n i t i a lv a l u e by t h e 8 t h d a y , when i t a g a i n f e l l t o a m i n i -mum ( F i g . 1 0 ) . At t h i s s e c o n d minimum, p e n i c i l l i nc o n c e n t r a t i o n r e a c h e d i t s p e a k .I n E x p . 8 a s e r i e s o f m o d i f i e d m e d i a w e r e madeup c o n t a i n i n g f r o m 0 0 4 t o 1 0 0 m g . P / m l . T h e s em e d i a d i f f e r e d f r o m t h e u s u a l CSL medium i n t h a tt h e y w e r e made up w i t h s p e c i a l l y t r e a t e d c o r n -s t e e p l i q u o r f r o m w h i c h m o s t o f t h e p h o s p h a t e hadb e e n removed by p r e c i p i t a t i n g w i t h p r e c i p i t a t e dc h a l k . G r a d e d a m o u n t s o f p h o s p h a t e a s KH2PO4w e r e a d d e d t o g i v e v a l u e s b e t w e e n t h e a b o v e e x -t r e m e s o f p h o s p h a t e c o n t e n t .B e t w e e n t h e s e l i m i t s t h e r e was no c o n n e x i o nb e t w e e n p h o s p h a t e c o n t e n t o f t h e medium andp e n i c i l l i n y i e l d . A l l y i e l d s o f p e n i c i l l i n w e r e n o r m a l

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F i g . 1 1 . S u l p h u r m e t a b o l i s m o f P . n o t a t u m . E x p . 1 0 ,s t r a i n 1 2 4 9 , m o d i f i e d CSL m e d i u m . o = t o t a l s u l p h u r i nm e d i u m ; * = p e n i c i l l i n t i t r e .t h e b u t a n o l s o l u b l e f r a c t i o n b e i n g r e j e c t e d . Th ea q u e o u s r e s i d u e c o n t a i n e d a l l t h e i n o r g a n i c c o n -s t i t u e n t s o f n o r m a l C S L , s o t h e r e was no n e c e s s i t yt o s u p p l e m e n t t h e me dium w i t h m e t a l l i c s a l t s .Th e t o t a l s u l p h u r and p e n i c i l l i n c o n t e n t s o f t h emedium w e r e d e t e r m i n e d a t 2 4 h r . i n t e r v a l s ( F i g . 1 1 ) .S u l p h u r d i s a p p e a r e d f r o m t h e me dium r a p i d l y u n t i lt h e 8 t h d a y , when i t b e g a n t o r e a p p e a r and a l m o s tr e a c h e d i t s i n i t i a l c o n c e n t r a t i o n by t h e 1 4 t h d a y .

DISCUSSIONNo c l o s e c o n n e x i o n c o u l d b e e s t a b l i s h e d b e t w e e n t h eg e n e r a l m e t a b o l i s m o f t h e m e t a b o l i t e s i n v e s t i g a t e d

3 4 2 1 9 4 6

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V o l . 4 0 METABOLISM OF PENICILLIUM NOTATUM 3 4 3and p e n i c i l l i n p r o d u c t i o n , e x c e p t i n s o f a r a s t h e d i s -a p p e a r a n c e o f t h e s e f r a c t i o n s g a v e an i n d i c a t i o n o ft h e g e n e ra l s t a t e o f mould d e v e l o p m e n t . The o u t p u to f p e n i c i l l i n by t h e mould f o l l o w e d t h e u p t a k e o fc a rb o n , n i t r o g e n and s u l p h u r by t h e m y c e l i u m w i t ha l a g o f 2 - 3 d a y s . Th e r e a p p e a r a n c e o f s u l p h u r andn i t r o g e n i n t h e m edi u m and t h e d e c l i n e i n m y c e l i a lc a r b o n i s p r o b a b l y r e l a t e d t o s e n e s c e n c e i n t h e o l d e rp a r t s o f t h e m y c e l i u m , b u t f u r t h e r i n v e s t i g a t i o n i sn e c e s s a r y . Th e p e c u l i a r f l u c t u a t i o n s i n p h o s p h a t ec o n t e n t o f t h e m edi u m a r e a s y e t i n e x p l i c a b l e . T h i sphenomenon m a y - b e c o n n e c t e d w i t h t h e o b s e r v a t i o nmade by t h e a u t h o r t h a t t h e a d d i t i o n o f p h o s p h a t eb u f f e r t o g r o w i n g c u l t u r e s o f P e n i c i l l i u m n o t a t u mp r o d u c e s o n l y a t r a n s i e n t c h a n g e i n t h e pH v a l u ew h i c h r a p i d l y r e v e r t s t o t h a t o f t h e p a r a l l e l c o n t r o lc u l t u r e . Th e i n h i b i t i o n e x p e r i m e n t m a y b e c o n t r a s t e dw i t h r e s u l t s o b t a i n e d by Mann ( 1 9 4 3 ) . T h i s w o r k e rf o u n d t h a t g o d i u m i o d o a c e t a t e ( M x 1 0 - 3 ) , s o d i u ma z i d e ( M x 1 o - 3 ) o r s o d i u m f l u o r i d e ( 5 M x 1 0 - 3 ) , whena d d e d t o c u l t u r e s o f A 8 p e r g i l l u s n i g e r on mediumc o n t a i n i n g 1 0 % g l u c o s e and i n o r g a n i c s a l t s , c a u s e da c o m p l e t e i n h i b i t i o n o f t h e p h o s p h o r u s m e t a b o l i s ma n d b r o u g h t r e s p i r a t i o n t o a s t a n d s t i l l .

SUMMARY1 . The f o l l o w i n g m e t a b o l i c f r a c t i o n s h a v e b e e nd e t e r m i n e d t h r o u g h o u t t h e g r o w t h o f P e n i c i l l i u ? mn o t a t u m : t o t a l c a r b o n , r e d u c i n g s u g a r , t o t a l n i t r o -

g e n , a m i n o - n i t r o g e n , n i t r a t e n i t r o g e n , v o l a t i l e b a s e ,p h o s p h a t e p h o s p h o r u s and t o t a l s u l p h u r o f t h em e d i u m , t o t a l c a r b o n and n i t r o g e n o f t h e m y c e l i u m ,and t h e r a t e and q u a n t i t y o f C 0 2 e v o l v e d by t h eg r o w i n g c u l t u r e .2 . P e n i c i l l i n p r o d u c t i o n by P . n o t a t u m f o l l o w e dt h e g e n e r a l a b s o r p t i o n o f c a r b o n , n i t r o g e n and s u l -p h u r i n t o t h e mould m y c e l i u m .3 . No s h a r p d i s c o n t i n u i t i e s i n t h e m e t a b o l i s m o fP . n o t a t u m h a v e b e e n o b s e r v e d .4 . The e x c h a n g e o f p h o s p h o r u s b e t w e e n t h emould m y c e l i u m and t h e medium f o l l o w e d a f l u c -t u a t i n g c o u r s e w h i c h i s a s y e t n o t u n d e r s t o o d .5 . Th e d e c l i n e i n t h e p e n i c i l l i n a c t i v i t y o f t h ec u l t u r e s was a c c e l e r a t e d by t h e a d d i t i o n o f a m m o -n i u m n i o n a l t h o u g h no pH c h a n g e was p r o d u c e d byt h i s t r e a t m e n t .My t h a n k s a r e d u e t o m y a s s i s t a n t s , Mr M . F i s h e r a n dM i s s F . G i l d e r , f o r c a r r y i n g o u t m u c h o f t h e c h e m i c a l a n a l y s i s .

REFERENCESA b r a h a m , E . P . , C h a i n , E . , F l e t c h e r , C . M . , F l o r e y , H . W . ,G a r d n e r , A . D . , H e a t l e y , N . G . & J e n n i n g s , M . A . ( 1 9 4 1 ) .L a n c e t , i i , 1 7 7 .C h a i n , E . , F l o r e y , H . W . , G a r d n e r , A . D . , H e a t l e y , N . G . ,J e n n i n g s , M . A . , O r r - E w i n g , J . & S a n d e r s , A . G . ( 1 9 4 1 ) .

L a n c e t , i i , 2 2 6 .C l a y t o n , J . C . , H a n e s , B . A . , R o b i n s o n , F . A . , A n d r e w s ,R . D . & H u n w i c k e , R . F . ( 1 9 4 4 ) . B i o c h e m . J . 3 8 , 4 5 2 .F o s t e r , J . W. & W o o d r u f f , H . B . ( 1 9 4 3 ) . J . b i o l . C h e m .1 4 8 , 7 2 8 .H o c k e n h u l l , D . J . D . & H e r b e r t , D . ( 1 9 4 5 ) . B i o c h e m . J . 3 9 ,1 0 2 .

L u k e , C . ( 1 9 4 3 ) . I n d u s t r . E n g n g C h e m . ( A n a l . e d . ) , 1 5 , 6 0 2 .M c C r e a d y , R . M . & H a s s i d , W. Z . ( 1 9 4 2 ) . I n d u 8 t r . E n g n gC h e m . ( A n a l . e d . ) , 1 4 , 5 2 5 .M a n n , T . ( 1 9 4 3 ) . N a t u r e , L o n d . , 1 5 1 , 6 1 9 .P e a r c e , P . H . & R o b e r t s o n , E . C . ( 1 9 3 6 ) . C h e m . & I n d . 5 5 ,5 4 3 .P e p k o w i t z , L . . P . & S h i v e , J , W. ( 1 9 4 2 ) . I n d u s t r . E n g n gC h e m . ( A n a l . e d . ) , 1 4 , 9 1 4 .P o p e , C . G . & S t e v e n s , M . F . ( 1 9 3 9 ) . B i o c h e m . J . 3 3 , 1 0 7 0 .S o m o g y i , M . ( 1 9 3 7 ) . J . b i o l . C h e m . 1 1 7 , 7 7 1 .Y o u n g b e r g , G . E . & Y o u n gb e rg , M . V . ( 1 9 3 0 ) . J . L a b . c l i n .M e d . 1 6 , 1 5 8 .

S t u d i e s on t h e S p e c i f i c i t y o f C h o l i n e E s t e r a s e i n H e l i x pomatiaBY K . - B . AUGUSTINSSON, D e p a r t m e n t o f C h e m i s t r y , R o y a l V e t e r i n a r y I n s t i t u t e , S t o c k h o l m

( R e c e i v e d 7 F e b r u a r y 1 9 4 6 )S i n c e 1 9 4 0 t h e p r o b l e m o f t h e s p e c i f i c i t y o f c h o l i n -e s t e r a s e h a s b e e n p l a c e d i n a n e w l i g h t , w h e n i i t wasshown by A l l e s & Hawes ( 1 9 4 0 ) and Hawes & A l l e s( 1 9 4 1 ) t h a t t h e e n z y m e s i n h u m a n b l o o d s e r u m andr e d b l o o d c e l l s h a d d i f f e r e n t p r o p e r t i e s . A m o n go t h e r t h i n g s , t h e s e a u t h o r s f o u n d t h a t e x c e s s a c e t y l -c h o l i n e d e p r e s s e d t h e a c t i v i t y o f t h e e r y t h r o c y t eenzyme b u t n o t ' t h a t o f t h e serum e n z y m e . T h e s eo b s e r v a t i o n s w e r e s u b s e q u e n t l y c o n f i r m e d i n

d i f f e r e n t q u a r t e r s . A c c o r d i n g t o R i c h t e r & C r o f t( 1 9 4 2 ) c h o l i n e s t e r a s e i n t h e e r y t h r o c y t e s i s d i f f e r e n tf r o m t h a t i n t h e b l o o d s e r u m , b e i n g e x c l u s i v e l ys p e c i f i c t o w a r d c h o l i n e e s t e r s and b e i n g a f f e c t e dd i f f e r e n t l y by c e r t a i n s u b s t a n c e s . S i m i l a r r e s u l t sw d r e a l s o o b t a i n e d by M e n d e l & Rudney ( 1 9 4 3 a ) ,a c c o r d i n g t o w h o m i t i s ' c l e a r b e y o n d d o u b t t h a tt w o d i s t i n c t c h o l i n e s t e r a s e s e x i s t i n t h e a n i m a lb o d y ' ( 1 9 4 4 ) . ' Z e l l e r & B i s s e g g e r ( 1 9 4 3 ) f o u n d t h e i r